重金属汞酶联免疫检测方法的研究

发布时间：2019-03-03 16:08

【摘要】：本实验建立了重金属汞离子的间接竞争酶联免疫(IC-ELISA)分析方法。由于汞离子不具有免疫原性,因此以异硫氰酸苄基乙胺四乙酸(ITCBE)为双功能螫合剂连接汞离子和钥孔血蓝蛋白(KLH)制备免疫原,在此基础上免疫新西兰大白兔获得特异性抗体,抗体效价为1：70000。用获得的抗体建立汞的间接竞争酶联免疫分析方法。本实验在优化了包被抗原包被量、抗体稀释倍数、稀释液离子强度、稀释液pH值和封闭液的基础上,建立了重金属汞间接竞争ELISA的抑制率标准曲线。建立的IC-ELISA检测方法的检测限为0.45μg/L,灵敏度为4.10μg/L。特异性实验表明,抗体对镍和铜有一定的交叉反应,交叉反应率分别为10.8%和13.5%,与其他金属均无明显交叉反应。本实验选取自来水、海河水、白芷、胡萝卜和虾喤为检测样本,通过基质影响消除实验确定样品前处理方法,同时进行添加回收实验。自来水和海河水过0.22μm水膜后用双蒸水稀释即可消除基质影响,白芷、胡萝卜和虾蛄采用硝酸和高氯酸体积比为5:1的硝化体系进行硝化处理,然后用双蒸水稀释可以消除基质影响。添加回收实验表明,自来水、海河水、白芷、胡萝卜和虾蛄中汞的回收率均在70%-120%,变异系数在20%以内。使用电感耦合等离子体质谱(ICP-MS)法验证方法的准确性,仪器检测结果和建立的ELISA分析方法具有很好的相关性(白芷样品检测判定系数为0.988),表明建立的方法具有很好的准确性。本实验建立的用于检测食品和药品中的重金属汞残留的酶联免疫检测方法,具有灵敏度高、使用简便、不需要昂贵的仪器等优点,可以作为批量样品的筛选方法和现场检测的便捷工具,对于应对突发食品或者药品汞安全事故提供了更好的选择
[Abstract]:An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) method for the determination of heavy metal mercury ions was established. Because the mercury ion has no immunogenicity, the mercury ion and keyhole hemocyanin (KLH) were ligated with (ITCBE) as the bifunctional chelator to prepare the immunogen, and the immunogen was prepared by linking the mercury ion with the keyhole hemocyanin (KLH) as the bifunctional chelator. On the basis of this, the specific antibody was obtained by immunizing New Zealand white rabbits, and the titer of antibody was 1? 700 000. The indirect competitive enzyme-linked immunosorbent assay (Elisa) for mercury was established by using the obtained antibodies. On the basis of optimizing the amount of coated antigen, the dilution multiple of antibody, the ionic strength of diluent, the pH value of diluent and the blocking solution, the standard curve of inhibition rate for indirect competition of ELISA by heavy metal mercury was established. The detection limit was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. the detection limit of IC-ELISA was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. The specific test showed that the antibody had some cross-reaction to nickel and copper, the cross-reaction rates were 10.8% and 13.5%, respectively, and there was no obvious cross-reaction between the antibody and other metals. In this experiment, tap water, Haihe water, Angelica dahurica, carrots and prawns were selected as samples. The method of sample pretreatment was determined by matrix effect elimination experiment, and the experiment of adding and recovering was carried out at the same time. After water film of 0.22 渭 m in tap water and water in Haihe River, the effect of substrate can be eliminated by dilution with double evaporated water. Angelica dahurica, carrots and oratosquilla were nitrified by nitrification system with the volume ratio of nitric acid and perchloric acid being 5:1. Then the effect of matrix can be eliminated by diluting with double evaporated water. The recovery rate of mercury in tap water, Haihe water, Angelica dahurica, carrots and Oratosquilla was 70% and 120% respectively, and the coefficient of variation was less than 20%. Inductively coupled plasma mass spectrometry (ICP-MS) was used to verify the accuracy of the method. The results of the instrument had good correlation with the established ELISA analysis method (the determination coefficient of the samples of Angelica dahurica was 0.988). It is shown that the established method has good accuracy. The enzyme-linked immunosorbent assay established in this experiment for the determination of heavy metal mercury residues in food and medicine has the advantages of high sensitivity, simple use and no need of expensive instruments, etc. It can be used as a screening method for batch samples and a convenient tool for on-the-spot detection, which provides a better choice for dealing with sudden mercury accidents in food or drugs.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.51

【参考文献】